1. Field of the Invention
The present invention relates to a class of minisatellite DNA called xe2x80x9ccomplex tandem repeatsxe2x80x9d (CTRs), which are of particular benefit in DNA typing applications. The present invention is also directed to a process of DNA typing using multiplex amplification comprising highly polymorphic complex and simple tandem repeat loci. The present invention is useful in analyses including, but not limited to, parentage, forensic, tissue origin, sample origin and genetic relatedness studies.
2. Description of the Background
Short tandem repeat (STR) polymorphisms are commonly used in DNA identification procedures, either as adjuncts to other genetic tests, or as stand-alone tests. Typically, when STRs are used for purposes relating to human identification, they are amplified by a process referred to as multiplex DNA amplification, which comprises amplification of two or more loci in a single reaction. Generally, the resulting amplified fragments are separated by size by polyacrylamide gel electrophoresis (PAGE) for subsequent analysis. The polymorphisms are then typed by determining their size in comparison to either similarly labeled known external standards or differently labeled internal standards. A DNA typing method of this nature, employing simple tandem repeats and PCR amplification, is disclosed in U.S. Pat. No. 5,364,759 (Caskey).
A critical parameter to evaluate when performing DNA typing for paternity analysis is the power of exclusion of the DNA typing method or test. Power of exclusion is the ability of a test to exclude false positive results that may lead to, for example, incorrectly accusing a man in a paternity test. The average or expected power of exclusion can be estimated using gene frequency distributions of systems in Hardy-Weinberg equilibrium (Brenner, C. and Morris, J. W., Paternity Index Calculations in Single Locus Hyper-Variable DNA Probes: Validation and Other Studies, Proceedings for The International Symposium on Human Identification, pp.21-76 (1989)), or by computing methods (Chakravarti, R. et al., Exclusion of Paternity: State of the Art. Am. J. Hu. Genet., 26:477-488 (1974); Garber, R. A. and Morris, J. W., General Equations for the Average Power of Exclusion for Genetic Systems of n Codominant Alleles in One-Parent and No-Parent Cases of Disputed Parentage, in Inclusion Probabilities in Parentage Testing, pp. 277-280 (1983); Chakravarti, A. and Li, C. C., The Effect of Linkage on Paternity Calculations, in Inclusion Probabilities in Parentage Testing, pp. 411-422 (1983)).
The exclusion probabilities of commonly used STR multiplexes are generally in the range of 85-91% for paternity analysis. Typically, at least three STR triplex analyses must be combined to provide a sufficient power of exclusion for most paternity analyses. For example, Alford et al. describe a battery of nine STR loci amplified in three triplexes which yields an exclusion power of 99.75% in Caucasians (Alford, R. L. et al., Rapid and Efficient Resolution of Parentage by Amplification of Short Tandem Repeats, Am. J. Hum. Genet., 55:190-195 (1994)). For comparison, the use of restriction fragment length polymorphic (RFLP) loci generally requires the testing of just one locus to produce the power of exclusion values equivalent to those obtained by analysis of three STR. RFLP analyses do, however, require larger amounts of DNA and time.
The low exclusion probabilities of commonly used STR loci are the most negative aspect of using STRs for paternity testing. The low exclusion probabilities of STR systems do not pose a serious problem in forensic testing in which the frequencies of both alleles of an individual are included in calculating match probabilities. However, in parentage testing only the frequency of the allele shared by the child and alleged parent is used for the probability calculation. Thus, although DNA typing with STR loci is simpler and faster to perform than RFLP and requires smaller quantities of DNA, many laboratories are reluctant to make the switch because of the sacrifice in exclusion power and accuracy.
Another disadvantage of the current STR multiplex DNA typing systems is that the amplification is rarely, if ever, clean. In other words, there is a considerable production of spurious bands thought to be a result of DNA polymerase slippage and mis-priming (see e.g., Tautz D., Hypervariability of Simple Sequences as a General Source for Polymorphic DNA Markers, Nuc. Acids Res., 17(16) 6463-70 (1989)).
These and other disadvantages are overcome by the methods of the present invention directed to DNA typing by multiplex amplification of highly polymorphic microsatellite loci.
In accordance with the present invention, powerful methods useful for DNA typing are provided. One embodiment of the present invention is directed to a method of multiplex amplifying comprising aplurality of loci, wherein at least one locus of the plurality is selected from the D3S2387, D4S2366, D5S1719 and D7S1804 loci of DNA. The plurality may further comprise the D18S535, D22S683 and D9S302 loci.
Another embodiment of the present invention is directed to a method of DNA typing comprising multiplex amplifying comprising two or more complex tandem repeat-containing loci referred to as xe2x80x9ccomplex tandem repeatsxe2x80x9d or CTRs. The CTR loci of the present invention may be selected from the group consisting of D9S302, D22S683, D3S2387, D4S2366, D5S1719 and D7S1804.
Another embodiment of the invention is directed to an allelic ladder useful in DNA typing. The ladders of the present invention may comprise at least 4 alleles from at least one locus selected from the group of loci consisting of D9S302, D18S535, D22S683, D3S2387, D4S2366, D5S1719 and D7S1804.
Another embodiment of the invention is directed to a composition comprising a plurality of amplified loci wherein at least two of said plurality are selected from the group consisting of D9S302, D22S683, D18S535. The plurality may further comprise the D3S2387, D4S2366, D5S1719 and D7S1804 loci. In a preferred embodiment, the plurality of loci of the composition comprises each of the D9S302, D22S683, D18S535, D3S2387, D4S2366, D5S1719 and D7S1804 loci.
Another embodiment of the present invention is directed to a kit for multiplex amplifying DNA comprising a plurality of loci wherein at least one locus of the plurality is selected from the group consisting of D3S2387, D4S2366, D5S1719 and D7S1804.